Ciência Rural,Gene Santa stacking Maria, as a strategy v.50:6, to e20190207, confer characteristics 2020 of agronomic importance in http://.doi.org/10.1590/0103-8478cr20190207 plants by genetic engineering. 1 ISSNe 1678-4596 BIOLOGY Gene stacking as a strategy to confer characteristics of agronomic importance in plants by genetic engineering Cássia Canzi Ceccon1* Andréia Caverzan1 Rogerio Margis2 José Roberto Salvadori1 Magali Ferrari Grando1 1Faculdade de Agronomia e Medicina Veterinária, Universidade de Passo Fundo (UPF), 99052-900, Passo Fundo, RS, Brasil. E-mail: [email protected]. *Corresponding author. 2Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brasil. ABSTRACT: Gene stacking refers to the introduction of two or more transgenes of agronomic interest in the same plant. The main methods for genetically engineering plants with gene stacking involve (i) the simultaneous introduction, by the co-transformation process, and (ii) the sequential introduction of genes using the re-transformation processes or the sexual crossing between separate transgenic events. In general, the choice of the best method varies according to the species of interest and the availability of genetic constructions and preexisting transgenic events. We also present here the use of minichromosome technology as a potential future gene stacking technology. The purpose of this review was to discuss aspects related to the methodology for gene stacking and trait stacking (a gene stacking strategy to combine characteristics of agronomical importance) by genetic engineering. In addition, we presented a list of crops and genes approved commercially that have been used in stacking strategies for combined characteristics and a discussion about the regulatory standards. An increased number of approved and released gene stacking events reached the market in the last decade. Initially, the most common combined characteristics were herbicide tolerance and insect resistance in soybean and maize. Recently, commercially available varieties were released combining these traits with drought tolerance in these commodities. New traits combinations are reaching the farmer’s fields, including higher quality, disease resistant and nutritional value improved. In other words, gene stacking is growing as a strategy to contribute to food safety and sustainability. Key words: co-transformation, re-transformation, sexual crossing, transgene, minichromosomes. Empilhamento gênico como estratégia para conferir características de importância agronômica em plantas via engenharia genética RESUMO: O empilhamento gênico se refere a introdução de dois ou mais transgenes de interesse agronômico na mesma planta. Os principais métodos de produção de plantas geneticamente modificadas com empilhamento gênico envolvem (i) a introdução simultânea, pelo processo de co-transformação, e (ii) a introdução sequencial de genes, pelos processos de re-transformação ou por cruzamento entre eventos transgênicos. Em geral, a escolha do melhor método varia de acordo com a espécie de interesse e a disponibilidade de construções genéticas e eventos transgênicos preexistentes. Também é apresentado aqui o uso da tecnologia de minicromossomos como tecnologia potencial de empilhamento gênico. O objetivo desta revisão é discutir aspectos relacionados à metodologia para o empilhamento de genes a combinação de características (obtida via empilhamento de genes de interesse agronômico) via engenharia genética. Além de discutir, é apresentado uma lista de culturas e genes aprovados comercialmente que tem sido usado em estratégias de empilhamento e uma discussão sobre normas regulatórias. Um número maior de eventos com empilhamento de genes foi aprovado e liberado no mercado na última década. Inicialmente, a combinação das características de tolerância a herbicidas e resistência a insetos era a mais popular, principalmente em soja e milho. Recentemente, estas características combinadas com tolerância a seca nessas culturas foram liberadas comercialmente. Novas características combinadas estão entrando na lavoura, incluindo aumento da qualidade, resistência a doenças e aumento do valor nutricional. Em outras palavras, o empilhamento gênico está crescendo como tecnologia para contribuir para a segurança alimentar e sustentabilidade. Palavras-chave: co-transformação, re-transformação, cruzamento, transgenes, minicromossomos. INTRODUCTION technology having the fastest adoption rate in relation to any other innovation in modern agriculture. In The use of genetically modified crops 2018, the global area cultivated with GMC was (GMC) presents numerous advantages to the 191.7 million hectares representing an increase of producer and to the consumer, as realized in greater approximately 113 fold since 1996, the first year productivity, reduced use of chemicals, cultivation GMC were commercially planted. The continued of plants in adverse environmental conditions and increase in GMC implementation in agriculture higher nutritional quality (RANI & USHA, 2013). resulted in economic and environmental benefits, The advantages offered by GMC resulted in this health improvement and social gains (ISAAA, 2018). Received 03.13.19 Approved 03.11.20 Returned by the author 04.07.20 CR-2019-0207.R2 Ciência Rural, v.50, n.6, 2020. 2 Ceccon et al. The first transgenic plants released (ZHU et al., 2017). Also, considerable progress has commercially were engineered for herbicide being made in molecular engineering in order to tolerance and this remains the predominant improve photosynthetic performance in C3 plants characteristic of transgenic crops. But, advances in by overproducing C4 photosynthetic enzymes. genetic transformation technologies, as well as the Many plants, including transgenic rice have been explosion of genomic sequencing technologies, have generated and intensively analyzed for this purpose facilitated the introduction of multiple genes and (TANIGUCHI et al., 2008). characteristics in a single variety using gene stacking The growing population and emerging strategies (LUNDRY et al., 2013). As a result, there environment challenges demand the development of has been an increase in the new development of more productive crops, more resistant to pests and crops with multiple genes in relation to those with diseases and tolerant to many stressful threats such single-gene engineered traits (LUNDRY et al., as high salt, drought, flood, freezing and adaptation 2013; ISAAA, 2019). to poor quality agricultural land. In addition, the Transgenic plants with combined need to save water in agriculture is compelling the characteristics (stacked traits) are those that have development of crops more efficient in its utilization. different characteristics in the same plant conferred The multigene engineering in plants can make those by different genes introduced by genetic engineering. goals more feasible to achieve. In 2018, GMC with stacked trait occupied 42% of the The purpose of this review was to global biotech crop area, and were more prevalent discuss the stacking of genes as a strategy to in the United States and Brazil (ISAAA, 2018). confer characteristics of agronomic importance in One example of stacked traits soybean is the event plants by genetic engineering. The aspects related MON87701 x MON89788, trade named Intacta™ to the methodology of production of plants with Roundup Ready™ 2 Pro, that carries the cp4 epsps gene stacking, events approved with combined gene, conferring glyphosate herbicide tolerance, characteristics and regulatory aspects are presented. and the cry1Ac gene, responsible for Lepidopteran resistance. In addition, plants are available with Methods for gene stacking stacked genes conferring resistance to different insect Gene stacking refers to the process of pests and/or tolerance to herbicides by different combining two or more genes of interest in the mechanisms. The most popular staked maize event genome of a single plant. There are different ways of in that category is the MON-89Ø34 x DAS-Ø15Ø7 obtaining plants with gene stacking. The procedures x MON-88Ø17 x DAS-59122, trade name Genuity® used can be separated into two main groups: (1) SmartStax™. This event harbor the cp4 epsps and simultaneous introduction methods; (2) sequential pat genes for glyphosate and glufosinate-ammonium introduction methods. herbicide tolerance and also carry the cry1Fa2, cry2Ab2 and cry1A.105 genes for Lepidopteran Simultaneous introduction methods insect resistance as well as the cry35Ab1, cry34Ab1 These methods refer to the introduction and cry3Bb1 genes for Coleopteran insects resistance of several genes in the same process of genetic (ISAAA, 2019). transformation. This technique is described as In addition, the genomic revolution has co-transformation and can be separated into two allowed the analysis of how the introduction of subgroups according to the methodology used: (1) groups of genes together affects biological systems. when all genes are present in the same plasmid it The multigene transfer technology allows researchers is called “co-transformation with single plasmid” to achieve goals that were once impossible, such as and (2) when genes are in separate plasmids it is importing a whole metabolic pathway and expressing called “co-transformation with multiple plasmids” entire multi-protein complexes (NAQVI et al., 2010). (FRANÇOIS et al., 2002). The major application of multigene transfer thus far Co-transformation with a single plasmid: has being the